A Single‐Molecule Perspective on the Role of Solvent Hydrogen Bonds in Protein Folding and Chemical Reactions

Dougan, Lorna; Ainavarapu, Sri Rama Koti; Genchev, Georgi Z.; Lu, Hui; Fernández, Julio M.

doi:10.1002/cphc.200800572

Cited by 41 publications

(47 citation statements)

References 81 publications

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“…We investigated this possibility by means of kinetic solvent isotope effects caused by replacing H 2 O by D 2 O in the samples. The stronger hydrogen bonding in D 2 O is thought to increase the hydrophobic effect and stabilize proteins (53)(54)(55)(56)(57). If water dominated the entropy change during the chaperone-mediated folding reaction, the kinetic solvent isotope effect in the chaperone should be significantly different from that of the autonomous folding reaction.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule spectroscopy of protein folding in a chaperonin cage

Hofmann

Hillger

Pfeil

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

113

124

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Molecular chaperones are known to be essential for avoiding protein aggregation in vivo, but it is still unclear how they affect protein folding mechanisms. We use single-molecule Förster resonance energy transfer to follow the folding of a protein inside the GroEL/GroES chaperonin cavity over a time range from milliseconds to hours. Our results show that confinement in the chaperonin decelerates the folding of the C-terminal domain in the substrate protein rhodanese, but leaves the folding rate of the N-terminal domain unaffected. Microfluidic mixing experiments indicate that strong interactions of the substrate with the cavity walls impede the folding process, but the folding hierarchy is preserved. Our results imply that no universal chaperonin mechanism exists. Rather, a competition between intra-and intermolecular interactions determines the folding rates and mechanisms of a substrate inside the GroEL/GroES cage.I n the recent past, a large number of components have been identified that control and modulate protein folding in vivo. This machinery includes molecular chaperones (1-3), sophisticated quality control systems, and complex mechanisms for protein translocation and degradation (3, 4), reflecting the importance of regulating the delicate balance of protein folding, misfolding, and aggregation in the cell. Such cellular factors exert conformational constraints on protein molecules that are expected to have a strong effect on the corresponding free-energy surfaces for folding (5). However, while the combination of cellular, biochemical, and structural data has led to some plausible qualitative models for the processes involved, mechanistic investigations comparable to those of autonomous protein folding in vitro (5-8) have been complicated by the complexity of the systems and the conformational heterogeneity involved (9). Even the autonomous folding of chaperone substrate proteins has been difficult to investigate because of their strong aggregation tendency (10). Contributions from confinement and crowding have been addressed in numerous studies using molecular simulations and theory (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), but many of these concepts have eluded experimental examination.Here, we take a step towards closing this gap by investigating the GroEL/GroES chaperonin (1-3, 9) with single-molecule fluorescence spectroscopy (21-24), a method that is starting to provide previously inaccessible information on chaperonemediated protein folding (25)(26)(27)(28)(29)(30). GroEL/GroES is a remarkable molecular machine that binds nonnative proteins and allows them to fold within a cavity formed by the heptameric rings of GroEL and GroES. However, the cavity is only slightly larger than the folded structure of typical proteins known to interact with the chaperonin. The large volume of unconfined unfolded protein chains compared to the size of the cavity raises the question of whether and how such strong confinement affects the folding reaction (12-16, 18, 31, 32). By labeling the classic substrate prote...

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Section: Resultsmentioning

confidence: 99%

Single-molecule spectroscopy of protein folding in a chaperonin cage

Hofmann

Hillger

Pfeil

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

113

124

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“…Using equation (2) we have predicted the pulling speed dependence of the unfolding force for six proteins from the literature, which have been studied at just one pulling 55 speed (Figure 9). The all-alpha-helical protein vascular cell adhesion molecule-1 (VCAM1) has previously been studied using AFM force extension at a pulling speed of 1000 nm/s, measuring an unfolding force of 40 pN 80 .…”

Section: mentioning

confidence: 99%

“…This entropy is reduced as the protein forms secondary or tertiary structures. Extending these native tertiary structures, to overcome the forces holding them together, has been achieved experimentally using a number of single molecule manipulation 55 techniques and requires forces of the order of piconewtons 17 . More than a decade ago, a pioneering study used an instrument called an atomic force microscope (AFM) to mechanically unravel a single molecule of the muscle protein titin 10 .…”

Section: Introductionmentioning

confidence: 99%

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Towards design principles for determining the mechanical stability of proteins

Hoffmann

Tych

Hughes

et al. 2013

Phys. Chem. Chem. Phys.

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The successful integration of proteins into bionanomaterials with specific and desired function requires an accurate understanding of their material properties. Two such important properties are their mechanical stability and malleability. While single molecule manipulation techniques 15 now routinely provide access to these, there is a need to move towards predictive tools that can rationally identify proteins with desired material properties. We provide a comprehensive review of the available experimental data on the single molecule characterisation of proteins using the 20 atomic force microscope. We uncover a number of empirical relationships between the measured mechanical stability of a protein and its malleability which provide a set of simple tools which might be employed to estimate properties of previously uncharacterised proteins. 25

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“…However, there are many instances where substitution with a heavier isotope results in an increase in the rate of a process, known as an inverse isotope effect, as observed in a variety of systems [17][18][19][20][21]. It has also been reported that hydrophobic effects are more pronounced in D 2 O compared with H 2 O [22,23] and the presence of D 2 O can have an effect on phase separation temperature of microemulsions [24], both of which would have an effect on the microphase separation of clusters during formation of RF gels. Hence, microstructure formation processes present in sol-gel systems, resulting from the interplay of chemical and physical processes, may be influenced by the mere presence of D 2 O, as well as the quantitative amount of D 2 O added to reaction mixtures, with the directionality of the effect also unknown; this may, therefore, limit the validity of insights about polymerisation and structure formation mechanisms obtained from previous NMR studies.…”

Section: Introductionmentioning

confidence: 99%

The impact of deuterium oxide on the properties of resorcinol-formaldehyde gels

Prostredny

Ballantine

Šefčı́k

et al. 2018

J Sol-Gel Sci Technol

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Resorcinol-formaldehyde gels offer a range of properties that can be exploited in a variety of applications, but better understanding of gel formation mechanisms is needed to enable rational control and optimisation of the physico-chemical characteristics of these materials. Our previous studies have focussed on investigating the formation pathways of these gels, using nuclear magnetic resonance and dynamic light-scattering techniques, as well as evaluating their final physical and chemical properties, via sorption and spectroscopic methods. Nuclear magnetic resonance has been used over the years to probe the chemical species formed during resorcinol-formaldehyde gel polymerisation, but the technique typically involves the prior addition of deuterium oxide to provide a deuterium lock for NMR measurements. However, the effect of deuterium oxide to resorcinol-formaldehyde systems is currently unknown, although the substitution of water by deuterium oxide has been previously reported to alter the chemical and physical properties of reacting systems. In this work we examine the effect of adding deuterium oxide to resorcinol-formaldehyde sol-gel synthesis at different dilution levels and the impact that this addition has on the final characteristics of the synthesised gels, in order to assess the validity of using NMR with a deuterium lock for the investigation of polymerisation mechanism in resorcinol-formaldehyde sol-gel processes. • High catalyst concentration systems less affected due to shorter gelation times• Low catalyst concentration systems more greatly affected due to weaker structure•

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A Single‐Molecule Perspective on the Role of Solvent Hydrogen Bonds in Protein Folding and Chemical Reactions

Cited by 41 publications

References 81 publications

Single-molecule spectroscopy of protein folding in a chaperonin cage

Single-molecule spectroscopy of protein folding in a chaperonin cage

Towards design principles for determining the mechanical stability of proteins

The impact of deuterium oxide on the properties of resorcinol-formaldehyde gels

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